Anti-interleukin (IL)-4 and -IL-5 antibodies downregulate IgE and eosinophilia in mice exposed to Aspergillus antigens

The effect of multiple divided doses compared with single-dose injections of antibodies to murine interleukin (IL)-4 and IL-5 in their respective downregulation of IgE and eosinophilia developing in a model of allergic aspergillosis is investigated. BALB/c mice were exposed to Aspergillus fumigatus antigens (Af) before and along with anticytokine antibodies. The kinetics of blood eosinophils, eosinophil peroxidase (EPO) in bone-marrow cells, scrum levels of IgE and Af-specific antibodies, Af-induced cytokine production and mRNA, and lung histology were studied. The results indicate that only multiple anti-IL-5 antibodies were effective in maintaining baseline levels of blood eosinophils. Multiple anti-IL-4 antibodies also downregulated eosinophils in the bone marrow, lung, and peripheral blood, although to a lesser extent than in anti-IL-5 antibody-injected mice. Significant correlation between the EPO activity and the eosinophil numbers in anticytokine antibody-treated mice was observed. The different anti-IL-4 antibody treatments downregulated IgE to the same extent. We conclude that multiple divided doses of anti-IL-5 antibodies arc required to sustain normal eosinophil levels in murine allergic aspergillosis. This information may be significant in the therapy of pulmonary allergic diseases.     

IgA is a more potent inducer of NADPH oxidase activation and degranulation in blood eosinophils than IgE

Human eosinophils can mediate both beneficial and detrimental responses in parasitic and allergic diseases. Binding of aggregated immunoglobulin to Fc receptors on eosinophils mediates important defence processes, including generation of activated oxygen species resulting from NADPH oxidase activation, and eosinophil peroxidase release following degranulation. The abilities of a matched set of IgA, IgG and IgE antibodies to elicit such responses in blood-derived eosinophils were compared using a chemiluminescence assay. IgA and IgG, but not IgE, were found to trigger NADPH oxidase activation and degranulation in eosinophils. This non-responsiveness to IgE did not result from receptor blockade by endogenous IgE since no blood-derived IgE was detectable on freshly isolated eosinophils. Moreover, while cross-linking of FcalphaRI by specific mAbs triggered NADPH oxidase activation and degranulation in blood-derived eosinophils, equivalent cross-linking of FcvarepsilonRI or FcvarepsilonRII did not elicit such responses. Therefore IgA is more potent at eliciting activated oxygen species release and degranulation in eosinophils than IgE, suggesting that the importance of IgA in eosinophil activation in immune defence and allergy may have been underestimated.     

Protective immunity induced in rat schistosomiasis by a single dose of the Sm28GST recombinant antigen: Effector mechanisms involving IgE and IgA antibodies

Rats immunized by a single dose of the recombinant Sm28GST antigen, using either aluminium hydroxide or Bacillus Calmette-GuSrin adjuvant, were significantly protected (up to 59% reduction in worm burden) against a challenge infection with Schistosoma mansoni cercariae. A follow-up study of the humoral response revealed the presence of high levels of IgE and IgA antibodies together with specific IgG. Sera from once Sm28GST-immunized rats induced a cytotoxic response for schistosomula targets in the presence of normal rat eosinophils, similar to the one induced by sera from twice immunized rats. Depletion or competition studies indicated the participation of both IgE and IgA antibodies in eosinophil-dependent cytotoxicity mechanisms. These results suggest the existence, in immunized rats exhibiting protection against schistosomiasis, of an original effector mechanism implying eosinophils and IgA antibodies, together with documented effector mechanisms involving IgE and eosinophils. In addition, they raise questions concerning the role of IgA antibodies in schistosomiasis.     

Eosinophil IgE receptor and CD23

In the present review, eosinophil Fc epsilon RII was compared to CD23, a differentiation marker of B cells. Biochemical analysis revealed that molecules of similar molecular weight were immunoprecipitated from eosinophils and B cells by an anti-CD23 monoclonal antibody (mAb) or by BB10, and anti-eosinophil Fc epsilon RII. By flow cytometry, a correlation was found between the binding of anti-CD23 mAb and myeloma IgE. However, a low expression of different epitopes of CD23 was observed in various hypereosinophilic patients. Northern blot analysis of eosinophil RNA with the cDNA probe of CD23 revealed a weak message in only 3 of the 6 patients expressing membrane CD23. The inhibition by anti-CD23 mAbs of IgE-mediated cytotoxicity and IgE binding to eosinophils clearly indicated the participation of CD23 or a related molecule in IgE-dependent eosinophil functions. However, the differential effects of anti-CD23 mAbs on eosinophils and B cells suggest major differences in the characteristics of the molecule expressed by eosinophils and by B cells.     

Role and modulation of CD16 expression on eosinophils by cytokines and immune complexes

BACKGROUND: Blood eosinophils express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFNgamma. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects. The present work is aimed at evaluating the possible mechanisms modulating eosinophil expression of CD16 and the biological functions of this receptor. METHODS: First, purified blood eosinophils were incubated with IL-1beta, IL-2, IL-4, IL-5, IL-9 or IL-16, GM-CSF, IFNgamma, eotaxin or 5-oxo-ETE and CD16 expression was measured. Second, the capacity of CD16 to mediate degranulation induced by IgG immune complexes (IC) was evaluated in eosinophils with low and high CD16 expression. Finally, serum allergen-specific IgE and IgG, and total IgE levels were measured at baseline in allergic asthmatics and correlated with changes observed in blood eosinophil CD16 expression (DeltaCD16) following allergen challenge. RESULTS: Only IFNgamma and IL-2 significantly increased the number of CD16+ eosinophils, respectively, 37 +/- 10% (p = 0.0038) and 38 +/- 8% (p = 0.0006), compared to control, 7 +/- 2%. IgG IC induced degranulation in eosinophils with low and high CD16 expression and monoclonal anti-CD16 and anti-CD32 antibodies inhibited this. IgG IC increased eosinophil CD16 expression (14 +/- 6%, p = 0.0008) and this effect was blocked by pretreatment with anti-CD32 antibodies. DeltaCD16 following allergen challenge correlated with the specific IgG/total IgE ratio (r(2) = 0.41, p = 0.036). CONCLUSION: These data suggest that formation of IgG IC is associated with surface eosinophil CD16 expression in asthma and that CD16 in cooperation with CD32 mediates IC-induced degranulation.     

Eosinophilic lung disease: immunological studies of blood and alveolar eosinophils.

Five patients with eosinophilic lung diseases and blood hypereosinophilia (PIE syndrome) were investigated clinically and by bronchoalveolar lavage (BAL). Comparative studies on blood and alveolar eosinophils were carried out after purification and selection of eosinophil subpopulations according to their density. A predominant 'hypodense' alveolar eosinophil population was found in BAL fluids of active chronic eosinophilic pneumonia (CEP). In addition, supernatants of alveolar macrophages obtained from CEP are able to enhance spontaneously the generation of eosinophil oxygen metabolites. Such eosinophil stimulation emphasizes a probable tissue cell cooperation. In addition, BAL permitted the study of membrane immunological markers on eosinophilic inflammatory cells endowed with migratory properties. An increase in eosinophils carrying surface IgE was demonstrated in alveolar cells from PIE Syndrome particularly with hypodense eosinophils from CEP patients. Although no specific stimulus is known at the present time, this work underlines the potential implication of IgE-mediated hypersensitivity processes in the pathogenesis of eosinophilic lung diseases.     

Plasma membrane antigens on light density and activated human blood eosinophils.

In order to study the membrane events which lead to human blood eosinophil activation and degranulation, six clone of mouse monoclonal antibodies were made to isolated blood eosinophils from a patient with the hypereosinophilic syndrome (HES). The antibodies were specific for the plasma membranes of blood eosinophils and neutrophils, eosinophil myelocytes and haemopoietic cell lines. A higher proportion of blood eosinophils from eight patients with the HES bound these antibodies compared to normal individuals. Five antibodies stained intermediate density eosinophils preferentially, and one stained the light density eosinophils most strongly. Normal blood eosinophils were induced to express more of these antigens either by culture alone (two antibodies) or culture with monocyte culture supernatant which activates eosinophils for increased cytotoxic capacity (four antibodies). It was concluded that several of the principal antigens on the eosinophil plasma membrane are also present on neutrophils and immature haemopoietic cells, and that their expression in mature blood eosinophils is related to the extent of eosinophil activation and degranulation. It is suggested that these antigens may be useful in studying the ways in which eosinophils alter their plasma membrane during activation and degranulation in vitro, and in the blood of patients with an eosinophilia.     

Regulation of surface FcepsilonRI expression on human eosinophils by IL-4 and IgE

BACKGROUND: Recent studies have demonstrated that eosinophils from allergic patients express low levels of FcepsilonRI on their surface, but the regulatory mechanisms of eosinophil surface FcepsilonRI expression are not fully understood. We investigated whether IL-4 and IgE, which are reported to regulate surface FcepsilonRI expression on human mast cells, are able to affect surface FcepsilonRI expression in normal human eosinophils. METHODS: Eosinophils purified from peripheral blood were cultured with IL-5 and with or without IL-4 and/or IgE, and surface FcepsilonRI expression was analyzed by flow cytometry using an anti-FcepsilonRI mAb, CRA-1. RESULTS: Apparent FcepsilonRI expression (approximately 1% of mast cell FcepsilonRI levels) was observed in eosinophils cultured with both IL-4 and IgE. A combination of IL-4 (>or=1 ng/ml) and IgE (>or= 0.5 microg/ml) was necessary for the maximal induction of surface FcepsilonRI expression. In the presence of IL-4 and IgE, eosinophils cultured for 2 days demonstrated low but statistically significant levels of surface FcepsilonRI, which reached a plateau after 7 days of culture. However, cross-linkage of surface FcepsilonRI molecules by CRA-1 or anti-IgE did not induce any eosinophil activation. CONCLUSIONS: IL-4 and IgE can affect the levels of surface FcepsilonRI on normal human eosinophils. FcepsilonRI expression on eosinophils may be regulated by a mechanism similar to that in mast cells.     

Total serum IgE levels in eosinophilic lung diseases]

BACKGROUND: We previously reported elevated levels of total serum IgE in patients with asthma, regardless of their atopic status. We hypothesized that certain factors inherent to asthma may contribute to this non-specific elevation of total serum IgE. In the current study, to evaluate the role of eosinophils in the regulation of total serum IgE, we examined whether peripheral blood eosinophil count is associated with total serum IgE level in patients with eosinophilic lung diseases. METHODS: Ninety-nine healthy controls, 277 patients with asthma, 15 patients with acute eosinophilic pneumonia, 21 patients with chronic eosinophilic pneumonia were studied for total serum IgE levels and peripheral blood eosinophil counts. RESULTS: Patients with acute or chronic eosinophilic pneumonia had significantly increased total serum IgE levels compared with healthy controls regardless as atopic status (p<0.001). In non-atopic subjects with eosinophilic lung diseases, total serum IgE level was significantly correlated with peripheral blood eosinophil count (r=0.42, p<0.001, n=57). CONCLUSION: Our findings suggest that, in addition to antigen-specific IgE production, non-specific IgE production may contribute to elevated levels of total serum IgE in patients with asthma or eosinophilic pneumonia. An increased number of activated eosinophils may underlie an increased total serum IgE level in these conditions.     

Eosinophil involvement in rheumatoid arthritis as reflected by elevated serum levels of eosinophil cationic protein.

Circulating levels of eosinophil cationic protein (ECP), an eosinophil specific granule protein, and numbers of peripheral eosinophils were determined in 42 patients with rheumatoid arthritis. At the time of the investigation the patients were without drug treatment. They had normal blood counts of eosinophils but on average a five-fold increase of the serum ECP values compared with healthy subjects. The intracellular content of ECP in eosinophils isolated from 14 patients was normal. High serum levels of ECP were particularly observed in patients with a disease of rather short duration but with a more aggressive course. Other factors associated with high ECP values were blood eosinophil counts in the upper normal range, high rheumatoid factor titre and increased inflammatory activity as defined by elevated serum haptoglobin and blood platelet counts. No relation was found between serum ECP and circulating immune complexes or serum total IgE. Synovial fluids obtained from 14 patients with rheumatoid arthritis contained very high concentration of ECP; on average nine times higher than those in the circulation of the patients. During corticosteroid but not NSAID therapy serum ECP decreased on average about 50% compared with pre-treatment values. Although eosinophils are not a notable feature of the synovial membrane infiltrate or cellular joint exudate, data obtained indirectly indicates their participation in the inflammatory reaction in RA.     

Intragastric Immunization of Rats withEntamoeba histolyticaTrophozoites Induces Cecal Mucosal IgE,Eosinophilic Infiltration,and Type I Hypersensitivity

We have investigated the role of IgE in the local immunity of intestinal amebiasis, a parasitic infection known to induce specific antibody-forming cells (AFC) and IgA antibodies in rodents and humans. We found that intragastric immunization of rats with glutaraldehyde-fixedEntamoeba histolyticatrophozoites significantly increased antiameba AFC in the Peyer's patches and spleen and that the lamina propria of the cecum from immunized animals was infiltrated by eosinophils armed with IgE antibodies. Morphometric analysis showed that IgE-containing cells and eosinophils were nearly three times more abundant in the cecum of immunized rats. Antigenic challenge with amebal lysates provoked an increase in the short-circuit current and in the transepithelial potential difference in Ussing-chambered cecum preparations from immunized rats. Although eosinophilia and the increase of IgE are common consequences of infection by parasitic worms, our results indicate that local immunity in intestinal amebiasis also involves IgE deposition, eosinophil infiltration, and type I hypersensitivity, which may explain some symptoms of amebic dysentery such as colic, abdominal tension, tenesmus, and bloody stools.     

IgE,Mast Cells,and Eosinophils in Atopic Dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease with specific immune and inflammatory mechanisms. Atopy is among the major features of the diagnosis criteria for AD but is not an essential feature. Thus, patients diagnosed with AD can be atopic or non-atopic. This review focuses on the role of IgE, mast cells, and eosinophils in the pathogenesis of AD. The known functions of IgE in allergic inflammation suggest that IgE and IgE-mediated mast cell and eosinophil activation contribute to AD, but direct evidence supporting this is scarce. The level of IgE (thus the degree of allergic sensitization) is associated with severity of AD and contributed by abnormality of skin barrier, a key feature of AD. The function of IgE in development of AD is supported by the beneficial effect of anti-IgE therapy in a number of clinical studies. The role of mast cells in AD is suggested by the increase in the mast cell number and mast cell activation in AD lesions and the association between mast cell activation and AD. It is further suggested by their role in mouse models of AD as well as by the effect of therapeutic agents for AD that can affect mast cells. The role of eosinophils in AD is suggested by the presence of eosinophilia in AD patients and eosinophil infiltrates in AD lesions. It is further supported by information that links AD to cytokines and chemokines associated with production, recruitment, and activation of eosinophils.     

Monoclonal antibodies to human eosinophil plasma membrane antigens enhance the secretion of eosinophil cationic protein.

This study was done to examine the nature of the membrane constituents involved in the secretion of eosinophil cationic protein (ECP) from human blood eosinophils. Three mouse monoclonal antibodies were used, which showed greater binding to membrane antigens on activated, and light density eosinophils from patients with an eosinophilia, than on nonactivated or normal density eosinophils. All three antibodies (EoN4, EoN5 & EoN6) stimulated normal density human eosinophils to secrete ECP, either alone or in association with sepharose-C3b. The antibodies bound to at least two separate sites on the membrane, which were distinct from the receptors for immunoglobulins, C3b, and eosinophil activating factor. One combination of antibodies increased the amount of ECP which was secreted. The membrane antigen recognized by antibody EoN4 was a glycoprotein, molecular weight 75 kD. These findings showed that ECP secretion may be induced by a wider range of stimuli than has been previously recognized, and that the antigens recognized by these monoclonal antibodies may play an important role in the induction of eosinophil degranulation.     

Enhancement of IgE-dependent eosinophil cytotoxicity to dinitrophenylated schistosomula by a nematode infection

In order to obtain direct evidence for the involvement of IgE in the eosinophil-mediated cytotoxicity, dinitrophenylated (DNP) schistosomula of Schistosoma japonicum coated with monoclonal IgE antibodies were examined as to whether they were killed by rat peritoneal eosinophils. The results indicated that the cytotoxicity of the eosinophils was dependent on a mouse monoclonal anti-DNP IgE antibody, but not on another monoclonal antibody specific to ovalbumin. Moreover, an enhanced cytotoxicity was observed when eosinophils were obtained from rats 4 weeks after infection with Nippostrongylus brasiliensis.     

Eosinophil membrane antigens: phenotypic frequencies in normal individuals and patients with the hypereosinophilic syndrome

The membrane antigen phenotype of eosinophils from six normal individuals and eight patients with the hypereosinophilic syndrome (HES) were examined to see whether subpopulations of eosinophils exist. Experiments were done with a panel of 6 monoclonal antibodies, using the fluorescent activated cell sorter, and immunocytochemistry. All six antibodies bound eosinophils and neutrophils, but not lymphocytes, monocytes, platelets or erythrocytes. The phenotypic frequencies of five of the six antibodies were increased in patients' eosinophils (p less than 0.005). This increase was associated with the intermediate-density eosinophils, while the antigen detected by antibody Eon 7 was associated with the light-density eosinophils. Normal eosinophils could be induced to increase their expression of these membrane antigens by incubation with mononuclear cell supernatants which are known to increase the cytotoxic capacities of eosinophil, 'activation'. It was concluded that there is a single eosinophil which undergoes post-mitotic differentiation in the blood, leading to activation and degranulation. The heterogeneity seen in patients' eosinophils reflect different stages of cell maturation and activation.     

The role of human mast cell-derived cytokines in eosinophil biology.

Eosinophil-mediated diseases, such as allergic asthma, eosinophilic fasciitis, and certain hypersensitivity pulmonary disorders, are characterized by eosinophil infiltration and tissue injury. Mast cells and T cells often colocalize to these areas. Recent data suggest that mast cells can contribute to eosinophil-mediated inflammatory responses. Activation of mast cells can occur by antigen and immunoglobulin E (IgE) via the high-affinity receptor (FcepsilonRI) for IgE. The liberation of proteases, leukotrienes, lipid mediators, and histamine can contribute to tissue inflammation and allow recruitment of eosinophils to tissue. In addition, the synthesis and expression of a plethora of cytokines and chemokines (such as granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-1 [IL-1], IL-3, IL-5, tumor necrosis factor-alpha [TNF-alpha], and the chemokines IL-8, regulated upon activation normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-1 [MCP-1], and eotaxin) by mast cells can influence eosinophil biology. Stem cell factor (SCF)-c-kit, cytokine-cytokine receptor, and chemokine-chemokine receptor (CCR3) interactions leading to nuclear factor kappaB (NF-kappaB), mitogen-activated protein kinase (MAPK) expression, and other signaling pathways can modulate eosinophil function. Eosinophil hematopoiesis, activation, survival, and elaboration of mediators can all be regulated thus by mast cells in tissue. Moreover, because eosinophils can secrete SCF, eosinophils can regulate mast cell function in a paracrine manner. This two-way interaction between eosinophils and mast cells can pave the way for chronic inflammatory responses in a variety of human diseases. This review summarizes this pivotal interaction between human mast cells and eosinophils.     

A new method for measuring eosinophil activating factors, based on the increased expression of CR3 alpha chain (CD11b) on the surface of activated eosinophils

The observation that activation of eosinophils in vitro with PAF increases the surface expression of the alpha chain of the complement receptor CR3 (CD11b) has been extended to other eosinophil activating factors. CD11b may be detected on activated eosinophils by reaction with mouse monoclonal anti-human CD11b IgG, following the addition of urease-conjugated sheep anti-mouse IgG. CD11b levels were increased on eosinophils after incubation with (a) recombinant colony stimulating factors, IL-3, GM-CSF and IL-5, at concentrations of 100 U/ml, or (b) with eosinophil activating factors, recombinant TNF alpha (1000 U/ml), EAF purified from mononuclear cell supernatants and PAF (10(-6) M). CD11b levels were not affected by IL-1 alpha, IL-2 or IFN-gamma. Unstimulated neutrophils had higher levels of CD11b than unstimulated eosinophils, but neutrophil CD11b was unaffected by IL-3, GM-CSF and IL-5 and was only slightly affected by TNF, EAF and PAF. Polyclonal rabbit antibodies to IL-3 and TNF neutralised their CD11b enhancing activities. The PAF antagonists WEB 2086 and WEB 2170 neutralised the CD11b enhancing activity of PAF. We conclude that measurement of CD11b expression on eosinophils is a convenient method for the assay of eosinophil activating activity.